CA2140891A1 - Peptides useful as internal standards for microsequencing and methods for their use - Google Patents

Abstract

The present invention provides a novel internal standard for amino acid sequencing which contains a peptide consisting of unnatural amino acid res-idues, such as ornithine, norvaline, norleucine and .alpha.-aminobutyric acid, that is capable of being sequenced si-multaneously with an unknown pep-tide or protein without interfering with the analysis. The internal standard peptide has an amino acid sequence containing at least two different unnat-ural amino acid residues having reten-tion times distinct from the corre-sponding retention times for natural amino acid residues. Information de-rived from the sequencing of the inter-nal standard allows determination of repetitive yield, lag, N-terminal block-age and discrimination between blank cycles caused by missed injection and blank cycles caused by faulty delivery of chemicals during the sequencer reactions. The present invention further provides novel synthetic control peptides containing from about 3 to 100 natural amino acid residues that are designed for use in monitoring the proper operation of amino acid se-quencers and to monitor peptide or protein cleavage reactions. The control peptide, or mixture of control peptides, are designed to obtain data for many or all common, uncommon and difficult to measure amino acids within 15 sequencer cycles and to pro-vide cleavage sites for at least 4 different amino acide cleavage reactants.

Description

21~0891 ''~VO 94/02856 PC~r/US93/06932 PEPTIDES USEFUL AS INTERNAL STANDARDS FOR MICROSEQUENCING
AND METHODS FOR THEIR USE
BAC K G R OlnND OF I~DE rNn~ENllO N
The present mvention relates to novel peptides useful in the amino acid seqllenrin~ cQnte~ and methods of their use. In particular, this invention S relates to an intern~l st~n~l~rd for amino acid sequencing comprising unn~tural amino acid res~ es that is capable of being sequenced simultaneously with an unknown peptide or protein without inte~ lg with the analysis. Further, this il,~,cl~ion relates to synthetic control peptides com~ illg natural amino acid re~ lues that are rlesi~en ~ for use in mo.~;lo.; -~ the proper operation of amino 10 acid seqlle-nrers and for co.~ri....;--~ that the system properly identifies all the cnmmon amino acid resi~lues. These synthetic control peptides can also be used as controls in a wide variety of ch~mic~l and enzymatic re~ct~iQnc to monitor cleavage and motlifir~tion re~rtion~

Amino acid sequenrers typically degrade a protein or peptide selectively and se~lçnti~lly into amino acid re-ci~ es, or deliv~lives of these res;~ s, that are capable of being qualitatively and q~ t;~ly identified.
For ;..~ e, the csmmnnly used Frlm~n sequential degr~lation involves the or~ ~c base catalyzed selective coupling of a peptide's N-termin~l amino acid 20 with phe~lylisothiocyanate. The del;v~ d amino acid is then cleaved from the peptide by tr~tment with a strong organic acid, typically as an anilinothi~nlinnn~ (ATZ) deliv~liv~. Repetitive coupling/cleavage cycles at the newly-fnrme~l N-lG....;..~1 amino acid left by the previous cycle provide for sequential s~alion of the amino acid resi~lues that form the primary structure 25 of the peptide. To determine the identity of the separated deliv~ s, the ATZ
dcliv~Liv~ is typically converted to a more stable phenylthiohydantoin (PTH) deliv~livti prior to analysis. These PTH derivatives can then be identified by a- variety of analytical procedures, such as by HPLC. The coupling/cleavage cycles, the PTH d~liv~t;~t;on procedures and the injection of the PIX derivatives onto 30 an HPLC can be ~ccomrli~he~l m~m~lly or, more commonly, by fully ~lltom~ted amino acid seql~ncers as described, for instance, in Applied Biosystems User Bulletin Issue No. 14 (November 18, 1985).

W094/028S6 ; PCr/US93/06932--9~ 2 Current intern~l standards available for use in an amino acid sequencer suffer from various disadvantages. For in~t~nre~ addition of a synthetic PTH amino acid derivative, such as PTH-norleucine, to one of the sequencer solvents is known. This type of internal standard, however, is capableS of in~lic~hng only that the sample was inje~cted properly onto the HPLC column.
A faulty injection step is only one of a ~ lfftu~le of possible m~lf~ln- tion~ that could occur during the sequ~nrin~ process. For in~t~n~e, the use of a PTH-amino acid intt~rn~l st~n~l~rd provides no inform~hon regarding whether the equipment is capable of actually sequçnring a s~mrle In ~-lrlihon, the PTH-10 norle~ ine deliv~liv~ is l~n~t~hle and must be added every 2-3 days, m~king qu~ ;rit ~I ;on very difficult and wasting ~ en~;vc; sequencer solvents.

R~lsch et al., BioPharm 2(5):40-43 (1989) ~ çlose the use of poly-I,-o- -;L~ . hydrochloride as an intern~l standard during automated protein 15 microsequencing. The poly-o...;Ll~ e molecule, which ranges in size from about 15,000 to 30,000 Daltons, degrades to provide a PTH deriv~tivt; that has a unique chromatographic retention time. Thus, obse~v~ce of the PTH-ornithine peak (or lack of the peak) for each sequencer cycle during the sequencing of an unknown protein provides inform~tion regarding illsLlulllent failure, "bad"
20 re~ent~ and sample-specific problems such as N-terminal blockage. However, because poly-o~ is a homopolymer, this intern~l st~ntl~rd is incapable of providing inform~hon regarding repetitive yield, which is an important index of sequencer pe. fiol "~n~e Further, because the PTH-ol ~l;L~ P. deliv~live is producefl in each sequencer cycle, this internal st~nrl~rd is extremely susceptible 25 to lag, or c~,yuvt;r from preceding cycles due to incomplete degr~dahon, ren-lerin~ quantiryillg the PTH-oll.ilhille peaks relatively me~ningless.
ition~lly, while the use of poly-ornithine as an internal standard provides some inform~tinn regarding instrument failure for a particular cycle or N-termin~l protein blockage, this internal standard is inc~r~hle of distinguishing30 between a blank cycle caused by a missed injection and a blank cycle caused by faulty delivery of chemicals during the sequencer reactions. Without this knowledge, the experiment would have to be repeated, which may not be ~ WO 94/02856 2 1 ~ 0 8 9 1 PCI/US93/06932 3 - . .; i;
possible for proteins that are only available in minute q~l~ntihes. Lastly, the poly-o...;l~.;,.e intern~l st~n~i~rd is more similar in size to a protein rather than a peptide and, thus, is not as easily washed from the sample support as a peptide s~mple Consequently, sequencer cnn-lition.c which provide for optimized 5 sequencing of the poly-o, ~IiLl~ e standard may not be a~ro~liate for sequen~ing an unknown peptidé.

Thus, there exists a need for an internal standard for amino acid sequen~ ing that does not interfere with the sequencing of an unknown y~otei 10 or peptide and can distin~ h between a blank sequencer cycle caused by the presence of mo-lifieri amino acids or m~r~hine m~lfilnrtion~, including blank cycles caused by missed injectionc and blank cycles caused by faulty delivery ofcllemir~lc during the sequencer re~t-on~. There also exists a need for an internal st~n~i~rd in which lag does not ilnLe~ere with subsequent 15 chromatographic peaks and which provides initial yield and several ac.;wate repetitive yields during the actual analysis of the sample unknown. Additionally, there exists a need for an intern~l st~n-l~rd having a molec~ r weight similar to peptides to provide a more accurate correlation when sequencing these components.
Mixtures cont~ining stable PTH amino acid re~ es, N,N'-diphenylthiourea (DPTU), dithiothreitol (DIT) and/or N,N-dimethyl-N'-phenylthiourea (DMPTU) have been used to optimize the sepalation conditions neerle-l for resolution of the PTH derivatives by the chosen analytical procedure, 25 such as by HPLC. However, these procedures opti,~ e only the final i~lenhfic~tion step rather than providing g li-1~nce for proper conditions throughout the repetitive coupling/cleavage/deriv~h7~hon/identific~hon cycles.
Proteins, such as ~-lactoglobulin, have also been used to verify the operation of the amino acid sequencer. However, o~l ;....,~hon of the sequencer using high 30 molecular weight components such as proteins can result in ina~propliate operating contlitiQns for sequencing lower molecular weight peptides, including contlihon~ which result in the peptide being "washed out" from the glass filter W O 94/02856 - ~ PC~r/US93/06932 -OQo9~ 4 - disc of the amino acid sequencer. Thus, even though ~-lactoglobulin cont~in~
a~ro~liate amino acid residues suitable for at least three determin~ht ns of therepetitive yield, these repetitive yield values may be inapplicable for peptide unknowns. Further, no single peptide is available that has even a few of the S uncommon or difficult to me~ure amino~cids suffi~ie~tly close to the N-telm.l,us to provide for sequencer op~ Qn that takes into account these rec;~lue while still providing inform~hon regarding the common amino acids.
Thus, there exists a need for a synthetic control peptide, or a mixture of synthetic conhrol peptides, capable of being used to monitor the proper 10 operation of an amino acid sequencer so as to allow op~;...i,~l;on of the sequencer with respect to the sequencing of peptides. In particular, there exists a need for control peptides ~le~iened to monitor the sequencing of the common amino acids as well as the rarely seen or difficult to measure amino acids in ~d~lihQn to providing an a~pL~liate residue s~hucture and sequence to allow 15 accurate determin~hon of lag and repetitive yield.

While it is possible to use e~i~hng proteins and peptides as controls for chemical and enzymatic reaction~, no polypeptide is available that is suitable for a wide variety of cleavages or reactions. Furthermore, because 20 proteins c~l~t~ many cleavage sites, use of proteins to monitor these reactions results in far too many fr~gment~, which yield complex chromatograms. Thus, mnl.;lu~ these re~ction~ by use of a control protein ~mnece~ss~rily complicates the subsequent analysis, m~king it liffic~-lt to determine the products and thereproducibility of the re~cti-)n. In ~ ition, the commercial ~l~palations of 25 proteil.s or peptides often vary in purity and some resi~lues may be modified in variable amounts in different preparations or from dirrerent m~nllf~Gturers.
Thus, there also exists a need for synthetic control peptides having amino acid sequences le~igned to have a limited but sufficient number of the approp-iate amino acid residues so as to allow the monitoring of a wide variety of chemical 30 and enzymatic reactions.

W O 94/02856 2 1 9 0 8 g I PC~r/US93/06932 S ~ ~
Throughout this applic~hQn, various public~honc are referenced.
The ~ closures of these publir~hQn~ in their entireties are hereby incorporated by lefercnce into this applic~hon in order to more fully describe the state of the art to which this invention pertains.
:
SUMMARY OF THE INVENTION

The present invention relates to novel peptides useful in the amino acid seqllen~ ing conte~l and methods of their use. In particular, this invention 10 relates to an amino acid sequencing internal st~ntl~rd peptide colllpl;sing nnn~tllral amino acid resitlues that is capable of being sequenced simultaneously with an unknown peptide or protein without i.,te-rel;..g with the analysis of the unknown peptide or ~roteill. Tnform~tion derived from the sequencing of the intern~l st~n~l~rd perTnit~ the mo~ ;..g of the sequencer performance during 15 the seqll~ncing of an unknown, including the determin~l~on of repetitive yield, lag and N-terminal blockage, as well as allowing for detection of and on between a blank cycle caused by a missed injection and a blank cycle caused by faulty delivery of ~h~mir ~l~ during the sequencer re~ction.c.

The internal st~nrl~rd co".p. ;~es a peptide con~i~ting essentially of nnn~ lral amino acid re~ les~ which has an amino acid sequence cont~ining at least two diLrere-lt llnn~hlral amino acid residues such that the retention timefor each unn~hlral amino acid residue following deli~ tion in an amino acid sequencer is distinct from the corresponding retention times for natural amino acid resi~hles. Two con~ec~ ve oc~;ullellces of at least one unn~tllral amino acid residue in the amino acid sequence are separated by at least one diLrelillg amino acid residue to allow deterrnin~tion of repetitive yield and at least 70% of thenn~ ral amino acid residues are positioned in the amino acid sequence so as to be separated by at least one differing amino acid residue to allow deterrnin~tion of lag.

W0 94/02856 ~ Pcr/uss3/o6932 2~40~9~ 6 Further, this invention relates to synthetic control peptides co..lL., ;~ natural amino acid residues that are ~le~i~ned for use in mo~ o~ ;.,g the proper operation of amino acid sequencers and for collLi~ g that the system plopelly identifir.s all the common amino acid residues. In one 5 embodiment, pairs of these control peptides may be sequenced simultaneously without data i~ .rerellce between each other ~r ~-lactoglobulin, a commonly used ~rot~ sequençing standard, for enough cycles to obtain data for common or unrommon amino acids, for easy or difficult to measure amino acids, and for initial and repetitive yields based upon only the stable and reliable PTH-10 deliv~Liv~s. In this manner, the control peptides provide a means to opli.lli~ethe sequencer for peptide sequencing or to .~imlllt~neously compare the sequencer perform~nce and o~ s~l;on contlihr)n~ for both proteins and peptides.

These synthetic control peptides can also be used as controls in a wide variety of chpmic~l and en~natic re~rtion~. Specific amino acid residues are ~llategically located to provide cleavage sites for various amino acid cleavage lC~ ;wt.c. Thus, the control peptides can be reacted with the cleavage reactantsand the res~llting fragments can be analyzed to qualitatively and qll~ntit~tively 20 assess the oc~;ul,ence, identity and extent of cleavage re~chr)n.~.

The control peptides of this invention co~llpiise from about 3 to about 100 natural amino acid residues and are ~lesi~ned to have 2 or more wlcon~ on or ~liffirlllt to measure residues within 15 amino acid resi~h~es from25 the N-te~lllillus of the peptide as well as at least 4 dirrelellt common amino acids within 15 amino acid residues from the N-tell.lil.us of the peptide.

DESCRIPTION OF THE FIGURES

Fig. 1 presents an HPLC chromatogram showing the retention times for the PTH derivatives of the following amino acids: D (aspartic acid), N(asparagine), CM-Cys (carbo7ymethylcycsteine), S (serine), Q (glutamine), T

~ WO 94/02856 2 1 4 0 8 9 I PCI/US93/06932 ~ ~"3 ~

(threonine), G (glycine), E (gl~lt~.,.ir acid), A (~l~nine), Y (tyrosine), Aab (cY-~minobutyric acid), P (proline), M (methionine), V (valine), Nval (norvaline), PE-Cys (pyridylethylcysteine), DPTU (N,N'-diphenylthiourea), W (tryptophan), Orn (o---;L1-;-~e), F (phenyl~l~nine), Ile (isoleucine), Lys (lysine), Leu (leucine) 5 and N-Leu (norleucine), Fig. 2 illu~ ates eY~mple pairs of res~ es, intlic~ted by the lines drawn between the individi~al amino acids, from which repetitive yield values can be dete~ ...;..ed when sequel~ring SEQ ID NO:1;
Fig. 3 shows a bar graph depiciting the cycle yields and repetitive yields for the B-lactoglobulin sequence with lag obtained from the simultaneous seqllenrin~ of B-lactoglobulin and SEQ ID NO:1;

Fig. 4 shows a bar graph depicting the cycle yields and repetitive yields for the intern~l st~nrl~rd SEQ ID NO:1 with lag obtained from me ~imult~neous sequen( ing of B-lactoglobulin and SEQ ID NO:1;

Fig. 5 shows a bar graph depicting the cycle sequence results for B-20 lactoglobulin and the inte.rn~l st~n~rd SEQ ID NO:1 obtained from thesimultaneous seq~le~ n~ of B-lactoglobulin and SEQ ID NO:1;

Fig. 6 shows three cycles each from two sets of HPLC
chromatograms obtained from the sequencing of SEQ ID NO:1 with unknown 25 sample to illustrate how an injection error is distinguished from a chemistry error during the sequencing of an unknown;

Fig. 7 in~lie~tes prefe~,ed pairs of residues used to calculate repetitive yield and ~lifficlllt amino acids within 10 resit1~les of the N-ter.~ ws for 30 the control peptides given by SEQ ID NO:3 and SEQ ID NO:4;

Fig. 8 shows the sequencing results from the simultaneous analysis of control peptides SEQ ID NO:3 and SEQ ID NO:4 for various sequencer cycles;

Figs. 9 and 10 show examples of the theoretical results obtained from the 5 reaction of some commonly employed chemical and enzymatic amino acid cleavage reactants with the control peptides having the sequences shown in SEQ
ID NO:3 and SEQ ID NO:4, respectively; and Fig. 11 shows the HPLC results for sequencing cycle 2 of SEQ ID NO:3 10 following reduction and alkylation of the cysteine residue with iodoacetic acid.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "natural amino acid" or "natural amino acid 15 residue" refers to the following naturally occurring amino acids or residues which occur in proteins: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, Iysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. The term "unnatural amino acid" or "unnatural amino acid residue" refers to either naturally 20 occurring amino acids or residues which are not included in the definition ofnatural amino acid, as defined herein, or non-naturally occurring amino acids orresidues, including both the D- and L-isomers, so long as the non-naturally occurring amino acid residues can be sequenced similarly to residues found in proteins or peptides. Many different unnatural amino acids exist; examples of 25 unnatural amino acids include, but are not limited to, a-aminobutyric acid, norleucine, norvaline and ornithine.

As used herein, the term "uncommon amino acid" or "uncommon amino acid residue" refers to the following natural amino acids or residues which do not 30 occur frequently or occur only moderately frequently in proteins: cysteine, g CA2 1 40891 (tryptophan, and histidine. Conversely, "common amino acids" or "common amino acid residues" refer to the other natural amino acids or residues listed above. An amino acid or amino acid residue is "difficult to measure" if the standard sequencing procedures produce a derivative of the residue that is reactive with 5 other components in the sequencer, unstable (i.e., decays into unmeasurable components before completion of HPLC analysis) or difficult to extract from the sequencing support or varies in rentention time (such that the HPLC peak becomesambiguous) due to changes in the HPLC buffers, which may change as they age.
Examples of difficult to measure residues include serine, threonine, histidine, 10 arginine, cysteine, and tryptophan.

The term "amino acid cleavage reactant" refers to a reactant that is capable of cleaving a protein or peptide predominately at a specific residue or at a specific sequence in the amino acid sequence based upon the identity of the amino acid 15 residues at the location. For instance, the amino acid cleavage reactant trypsin is capable of cleaving a protein or peptide at positions in the amino acid sequenceimmediately following a Iysine residue or an arginine residue, considering the sequence as ranging from the N-terminus to the C-terminus. Other examples of amino acid cleavage reactants include, but are not limited to Endoproteinase Asn-20 C, Endoproteinase Lys-C, Endoproteinase Arg-C, Endoproteinase Glu-C and Endoproteinase Asp-N, which can be obtained from Mannheim Boehringer Biochemica (Indianapolis, IN) and BNPS-skatole, trypsin, cyanogen bromide, V8-E-AB (V8 protease which cleaves E in ammonium bicarbonate), V8-DE-Po4 (V8 protease specific for D and E in phosphate buffer), formic acid and acetic acid.
INTERNAL STANDARD PEPTIDES
This invention provides for a synthetic, non-interfering internal standard for amino acid sequencing comprising a peptide consisting essentially of unnatural amino acid residues selected such that the retention time for each unnatural amino 30 acid residue following derivatization in an amino acid sequencer is distinct,preferably baseline resolved, from the retention times for corresponding naturalamino acid residues. It is important that the peptide does not contain any natural amino acid residues because their presence could W094/02856 ~ Pcr/us93/06932 ~
2~,40~9~ 10 ;..~e~relG with the analysis of the unknown sample. Further, the elution times for the deliv~liv~s, such as PTH-derivatives, of the selected nnn~tllral amino acid resid~les are within the times norm~lly seen for the corresponding natural amino acid derivatives formed using the same sequencer reactions. Because the 5 intern~l st~nt1~rd peptide can be sequenced simultaneously with an unknown peptide or ploLei,- sample in an amino acid sequencer without in~e,r~-;l,g with the analysis of the unknown, the intern~l standard provides a means to monitor the sequencer perform~n-~e during the sequencing of the unknown. Fig. 1 depicts a typical HPLC chromatogram showing the rentention times for PTH-10 de~ ives of natural amino acids and some unn~tural amino acids. The elutiontimes for the nnn~hlral amino acids are adequately difre~e,lt from those of the natural PTH-amino acids.

The amino acid sequence of the internal standard peptide is 15 ~e~ignPd such that multiple o~;ul-~nces of at least one particular residue that yields a stable and reliable PTH-derivative are sepalaled by at least one, prererably two or more, other residues so as to allow c~lcul~tion of initial andrepetitive yields for that residue.

FurthP-rmore, the amino acid sequence of the internal standard peptide is also designed such that at least 70%, preferably 80%, more preferably100% of the mllltiple oc~;wlellces of any particular residue are separated by atleast one, pleferably two or more, other resirlues. In the N-terminal portion ofthe peptide, it is piere-led that 100% of the multiple oc~ullcnces of any 2~ particular residue are separated by at least one, preferably two or more, other rec;~lues. In this manner, the lag caused by the proces.~in~ of the particular residue in an earlier amino acid sequencer cycle will be .ni"i",;,ed or elimin~ted during the subsequent processin~ of the same kind of residue in a later cycle.
Re~use positioning of charged residues near the C-te--l~hlus of the peptide may 30 promote adherence of the internal standard to the sample support in the sequencer, it may be useful to position resi(lues, such as ornithinç, in the C-te~ .s region such that they are not separated by other residues. However, a ~ WO 94/028~6 2 ~ 4 0 8 9 I PCI /US93/06932 r ; .
11 ' I,, single o.~ e at the C-terminus would mimic the type of peptide obtained by digesffon with hypsin and would allow for sequencing to the end of the peptide.

Although the number of re~idlles cont~ined in a single peptide 5 chain of the intern~l st~n-l~rd could be any number 2 or larger, cost and timecon~ erations will generally lim* the peptide size to between 2 and 100 res~ es, l,re~eial~ly between S and 60 resillues, most preferably between 10 and40 res;dues. Typically, the longer the peptide is, the less likely it is to "wash out"
of the amino acid sequencer. Thus, a higher molec~ r weight internal standard 10 tends to behave more simil~rly to a proteill, including having an increased repetitive yield, co...~ cd to lower molecular weight peptides. Decreasing the length of the intern~l st~nd~rd tends to have the opposite effect. Thus, pepffdes of varying len~th~ can be synth~si7e~l to provide internal st~nd~rds capable of more re~ h~lly mo..;lo~ the effect of the amino acid sequencing process on 15 similarly sized peptides.

Furthermore, the identity and location of hydrophobic and hydrophilic amino acid resitllles in the peptide can be le~i~nsd to avoid solubility problems and difficulty during the HPLC pllrific~hon For in~t~nce, 20 because of the hydrophobicity of norleucine residues, a peptide cont~inin~ too many of these reSi~lues can be lifflrlllt to dissolve and purify by HPLC. Thus,an excess of norleucine resitlues in the internal st~ntl~rd should be avoided Collve~ely, more hydrophilic residues, such as o. ..iLh;.~e, aid in solubility and subsequent pllrih~hon Incorporation of residues in the peptide that cause problems in the synthesis or deprotection of the peptide should be avoided to cil~;ul~lv~llL or decrease the problem of low yields for the final product. Furthermore, using an Applied Bio~y~;l.ls, Inc. (ABI) (Foster City, CA) 475A sequencer modified with 30 bottle and regulator updates with an on-line Model 120A PTH-amino acid analyzer, PTH-~-cyclohe7yl~l~nine did not have an HPLC retention time unique from the col-esponding PTH-common amino acids, and should not be Wo 94/02856 Pcr/Us93/06932 '--as~

incorpo~aled in internal standard peptides designed to functinn in this system. If desired, more expensive llnn~tural amino acids, such as ~2,4-diaminobutyric acid and L,2,3-~i~minopropionic acid, can be avoided.

Using the above guidelin~s, synthesis of various internal standard peptides from synthetic ~lnn~tural amino acids can be performed using standard procedures, such as t-boc chel~ y. However, the use of FastMoc~ chemistry is pr~feiled because cleavage of the peptide from the resin and deprotection is simpler than the HF needed for synthesis using t-boc chemi~l.y. In short, 10 intern~l st~r~d~rd peptides can be synthesi7ed by solid phase synthesis using an ABI Model 430A Peptide Srtheci7er. The FastMoc chel~ y approach (0.25 mmnl~r scale) can be uhli7P~1 es~e~h~lly as described in "FastMoc ~he-.,icL.y;
HBTU Activation in Peptide Synthesis on the Model 430A", Applied Bio~y~l~ms User Bulletin Issue No. 32 (November 1990) and the synth~si7~r can be 15 controlled by the HBTU.25 Run File of the ABI FastMoc~ soflwa~ (version 1.4). Of course, other run files and synthesi_ers may be succe~ lly used.

The intern~l standard may further co,.lplise a charged substrate or solid support attached to or near the C-telmil~us of the amino acid sequence so 20 as to ...i..;...;,e wash out of the intern~l st~ncl~rd from the amino acid sequencer.
By connecting the peptide to such m~teri~l~, or by synthesizing them onto materials, the effective molecul~r weight of the intern~l st~n~l~rd increases, le~-lin~ to effects similar to increasing the molecul~r weight. For instance, suitable substrates include but are not limited to the following substrates: a 2~ peptide co~ at least one charged llnn~tllral amino acid residue, a peptide com~ ing at least one charged natural amino acid residue and peptides comI7ri~ing a mixture of charged lmn~tllral and charged natural amino acid resitlues. Furthermore, the internal standard peptide can be synthesi7ed onto a mllltirl~ antigenic peptide resin, such as a t-boc MAP resin, Fmoc MAP Resin 30 4-Branch or Fmoc MAP Resin 8-Branch (obtained, for example, from ABI).
ition~lly, the peptide can be covalently attached to other solid supports, similar to those used in solid phase sequencing 21408gl WO 94/02856 ~ PCr/US93/06932 The intern~l st~n~rd of this invention can be used to monitQr the ye. r~ n~e of the amino acid sequencer during the sequencing of an unknown peptide or protein. A small amount, preferably close to the amount estimated for the unknown, of the internal st~ncl~rd is placed on the glass filter disc of an 5 amino acid sequencer (any m~nilf~ct~lrer for this type of instrument). The unknown sample to be sequenced is also placed on this filter and the experiment is started. The sequencer ~lltom~tlr~lly repeats a series of reactions on both the unknown sample and the internal st~n~l~rd. The general steps are as follows:
couple the N-termin~l amino acids with phenylisothiocyanate; cleave the amino 10 acids to yield the PTC derivatives, convert the PTCs to PTH deliv~tives, and inject the two PTHs into an HPLC system. The HPLC unit then separates the PTH deliv~livt;s, which are idenfffied by coml)~illg the retention times with those of known st~n~l~rds. Each cycle of the sequencer result should give the e~l-ecte~l synthetic PTH amino acid for the intern~l st~n~l~rd in an amount that15 is re~on~hle and reproducible when the equipment is opel,l~ing norm~lly.
Furth.ormore, each cycle of the sequencer can be checked to verify that no more than l~c~o.~hly expected lag for the synthetic unn~t~lral PTH amino acids is ~.~ se~,l.

An initial yield can be c~lcul~ted for the internal standard by c- ,--p~ the quantity of the synthetic amino acid that is obtained in cycle 1 (PTH-Orn for in~e.rn~l standards defined by SEQ ID NO:1 and SEQ ID NO:2) with the amount of the peptide that was added originally. This initial yield should norm~lly be about 40-60% for a sequencer that is f~mchoning properly.
Several repetitive yields from the internal standard can be c~lcul~ted during the actual sequencing of the unknown sample. The repetitive yield values are extremely important in detel ~ g the operation of the equirment during a run. For instance, the Applied Biosystems sequencer is guaranteed in service maintenance contracts to have a repetitive yield of at least 92%. This value is tested in an experiment using the ABI protein standard, ~-lactoglobulin. However, a good repetitive yield during the sequencing of this W094/02856 ~ PCr/USs3/06932--2~0~ 14 pLoLein does not insure that the equipment will work ~ropelly in the next ~A~e~ ;...Pnt For instance, any of the many (1OOs~ of valves, lines, and circuits plesel~t in the sequencer could develop problems or leaks at any time. Also, a~n~x;.-~tely 15 di~erellt re~gent~c are being contin~l~lly consumed and 5 replaced. Occ~ion~lly, some bottles of these chemir~lc are found to give poor results, but, prior to this invention, was not norm~lly realized until several unknown s~mplec had been analyzed. However, the internal standard of this invention allows the me?cl~rement of repetitive yield during the sequencing of the unknown. Thus, sequencer errors can be d~te.l..;.-ed as they occur, 10 ~ VCil~Lillg the waste of time and, frequently, ~ ecess~.y loss of unknown s~mple. The repetitive yield (RY) is determined by the following formula:

RY [pmoles P~ in c~cle y] tl/ (Y resld~e #-X resid~e ~)]
pmoles P7H in cycle X

Fig. 2 ill~ales some of the various possible pairs of recicl~les from which 1~ repetitive yield values can be calc~tl~ted when sequencing the internal standard SEQ ID NO:1 .cim~llt~neously with an unknown peptide or protein. These pairs are in(lic~ted by the lines drawn between the individual amino acids. For clarity, lines between various ornithin~ residues were omitted; o, .,ill.;"e residues can be used to determine repetitive yield.
' The intern~l standard of this invention is a more realistic standard for the sequencing of peptides than a protein internal standard because a protein st~n-1~rd is not as sensitive to being washed from the sample support asthe peptide sample being sequenced. Optimi7ing the sequencer flow rates, 25 re~ction ti-m-es~ etc. for the internal standard would also optimize the m~hine for internal peptides obtained enzyme digests or chemical cleavages.
Furthermore, o~ g the sequencer for the internal standard would also set the m~rhine correctly for the sequencing of proteins. Lags can be easily W O 94/02856 21 4 0 8 9 1 PC~r/US93/06932 i~ Q
detected for the intern~l st~nd~rd because the amino acid residues are separatedby at least one other residue.

The internal standard is capable of detecting errors attributable to 5 the unknown, such as N-terminal blockage in the unknown protein or peptide.
That is, cycles which do not produce HPLC peaks for the unknown but yield the e~ecte~l peaks for the intern~l standard allow the operator to conclude that theh~l~uent is fimt honin~ properly and the lack of peaks lies with the sample.
Furthermore, the intern~l st~nrl~rd can tiictin~lich between a missed injection 10 and a blank cycle caused by faulty delivery of chemicals during the sequencerre~ction.~. That is, the in~e.rn~l standard will yield at the next cycle either the residue e~ ectecl following an injection problem or the one that was expected inthe missed cycle. These results would in~ te that the previous amino acid was not injected or that the chemi~l re~ction~ did not occur during that cycle, 15 lc*,e~iliv~ly. For eY~mple, if a blank occurred at cycle 3 of the internal standard defined by SEQ ID NO:1, the next residue would be expected to be the following depending upon the problem:

correct sequence orn nvl NLE aab missed injection orn nvl - aab missed chemicals orn nvl - NLE

Preferably, the internal standard peptide either colllaills at least three diLrerclll lmn~ral amino acids or does not exist solely as altern~ting amino acid residues 25 so as to be able to differentiate between missed injection errors and m~ ln~tinning chemical proce~in~ errors when two adjacent cycles are blank.
For PY~mple, a sequence having the pattern ABABABABAB would not diLrelcllliate between these errors. However, if a third different residue is ~lese-~ in the sequence following the blank cycles, such as in the pattern 30 ABABABABAC, or if the pattern is not solely alternating, such as ABABABABAA, the internal standard peptide would be able to differentiate between these types of errors.

Wo 94/02856 - Pcr/uss3/o6932--~ ~ 40~9~ 16 An ~rlrlihon~l advantage of the internal standard peptide of this invention is that the PTH de~iv~tiv~s formed during sequencing may act as c~rriers for the PTH re~i~lues formed from the unknown sample. It would be possible to ~ rove the sequence results of, say, 1 pmol of an unknown by 5 adding 50 pmol of the intern~l standard~ ition~lly~ the internal standard peptide could act as a carrier to p~ ;nt commonly observed loss of unknown sample peptides during their purific~tiQn for sequencing. For example, 200 pmol of the standard could be added to tubes used to collect peptides during HPLC purific~hrlnc. The presence of the standard would not interfere with the 10 subsequent sequencing analysis.

The present internal standard is more particularly described in the followmg eY~mples which are intended as illu~Lla~ivt; only since numerous modifi~ ~hons and variations therein will be apparent to those skilled in the art.
Fxample l The peptide whose sequence is given by SEQ ID NO:1 was sy-nthçsi7ç~l by solid phase synthesis using an ABI Model 430A Peptide Synth~osi7çr and the FastMoc~ che~ Lly as described above. The following 20 synthetic amino acids, with the incliçated protecting groups, were use for the synthr.cis: 9-fluore"yl-"ethoAycall,onyl(Fmoc)-L,norleucine; Fmoc-norvaline;
Fmoc-o, .~iL~ e (t-butyloxcarbonyl) and Fmoc-L,a-aminobutyric acid. The first amino acid was attached to thep-llydlo,~y-methylphenoxymethyl-poly~lylelle (HMP) resin by the syntheci7er (cycles rfmclld, cfmc 11d, and afmc l1d) and 25 ~en capped with benzoic anhydride (cycles rfmcll, cfmc l1, and afmc l1). The other re~ ues were added using the HBTU.25 Run File, as liccussed above (cycles RHBTU.25, ~ 1 LJ.25, and AHBTIJ25X).

The N-terminal Fmoc group was autom~tic~lly removed by the 30 Synth~ci7çr using the RNH2.25, CEND, and AEND cycles. The internal standard peptide was then cleaved from the resin and simultaneously deprotected by incubating 0.2 g of the peptide-resin for 1.5 h in a solution WO 94/02856 2 1 4 0 8 9 I Pcr/us93/06932 composed of 0.75 g crystalline phenol, 0.25 ml 1,2-eth~ne-lithiol, 0.5 ml thio~ni.cole, 0.5 ml deionized water, and 10 ml trifluoracetic acid. The peptidewas then precif.i~ ed in ethyl ether, filtered, and washed on a fritted glass funnel. This procedure is described in the ABI booklet "Introduction to 5 Cleavage Techniques-Strategies in Peptide Syn~h~si~

The peptide was purified by dissolving it in 0.1% TFA and purified by high ~ n~e liquid chromatography (HPLC) utlli7ing an Aquapore RP-300 collumn equilibrated with 0.1% TFA. The sample was then eluted with a 7.5 10 min linear gradient of 0% to 52% aceto..;l~ ;le co.-l~;..;..g 0.07% TFA. The peptide whose sequence is given by SEQ ID NO:2 was synthesized similarly.

E~...y1~ 2 The ~lotein B-lactoglobulin (100 pmol) was sequenced 15 .~imlJlt~n~ously with SEQ ID NO:1 (100 pmol) on an ABI 475A sequencer morlifiet1 with bottle and regulator updates and equipped with an on-line Model 120A PTH-amino acid analyzer. Porton Peptide supports were utilized with the n~-rm~l cartridge. Figs. 3-5, show a bar graph depi~ the cycle yields and repetitive yields for the ~-lactoglobulin sequence with lag, the cycle yields and 20 repetitive yields for the internal s~n~l~rd (SEQ ID NO:1) with lag, and the sequence results for B-lactoglobulin and the intern~l standard (SEQ ID NO:1), respectively.

In the course of using SEQ ID NO:1 as an internal st~nd~rd when 25 sequencing various unknown proteins and peptides, various injection and che~ y errors were detected. Fig. 6 shows three cycles from two sets of HPLC chromatograms obtained from the sequencing of SEQ ID NO:1 with unknown s~mples to illustrate how an injection error can be distinguished from a che~ y error. In both sets, the cause behind the blank cycles was clearly 30 delinlo~te-l wo~ C A 2 1 4 08 91 PCI/US9:~/0693Z--CONIROL ~;~lll~ES

This invention also provides novel syn~hetic control peptides ~le-signe~l for use in monitorin~ the proper operation of amino acid sequencers 5 that have amino acid sequences containing from about 3 to about 100 natural amino acid reci~lue~ These control peptides, which plcLelably can be sequenced in an amino acid sequencer without data il~tel rG- ellce from the protein standard ctoglQbulin, are constructed to provide sequencing inform~hon quicldy to the ~el~tor so as to allow efficient op~-mi7~tion of the sequencer, particularly with 10 respect to the seq~enring of unknown peptides. In particular, the control peptides are ~le~igne-i to provide sequenring ;..rO....~tiOll regarding uncommonand rliffic~lt to me~llre amino acids as well as many, preferably all, common amino acids during the early cycles of the amino acid sequencer. Further, cr.mpo~it r~n~ co~ g at least two of these control peptides are speçific~lly 15 ~ igned to provide this sequencing inform~tion without data inte~ cllce with each other or, preferably, with ~-lactoglobulin.

In particular, the amino acid sequences for these control peptides are constructed so as to place at least some uncommon and/or difficult to 20 me~re amino acid re~i-lues within 15 amino acid resitl~ es from the N-tellllilli of the peptides. Fle~ldbly, the amino acid sequences have at least 2 ~mcommon or rliffic~~lt to measure resi(l~es, such as a cysteine residue, a tryptophan residue, a serine residue, a threonine residue, a histidine residue, an arginine residue or a methionine residue, located within 15 amino acid re~ es, more ~reLerdbly 25 within 10 amino acid resicl~es, from the N-tel...i....s of the peptide.
Furth~rmore, the rem~inin~ residues are selected so as to provide a wide varietyof common amino acids, preferably having at least 4, more preferably at least 7,diLCerellt common amino acids located within 15 amino acid residues of the N-te....i..~s. Prefelled peptides are constructed such that at least one residue, 30 more plefeldbly two different residues, which possesses a stable PTH-delivdtive is repeated at least one time, but not immediately adjacent the first oc~;ullcllce~
within 1~ amino acid residues of the N-te~ lus to allow c~lc~ tion of WO 94/02856 2 1 9 0 8 9 1 ; PCr/US9~6932 repetitive yield. Useful control peptides include, but are not limited to, peptides having me sequences given in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

S .A~ ihon~lly, me control peptides ~rcferably are designed so as to not il~lelrele with me ~im~llt~neous sequencing of ~-lactoglobulin. In this manner, me control peptides can be sequenced .~im~llt~neously with ~-lactoglobulin so as to provide inform~tinn COIIIp~illg the sequencing of the peptides with this ABI protein standard. To be non-intelrerci"g, each control 10 pepffde amino acid residue at a particular residue location number differs from the ~-lactoglobulin amino acid residue having a residue location number ranging at least from the particular residue loc~hon number minus 1 to the particular residue loc~hon number plus 1. In this co"lexl, the residue location number for either the control peptide or for ~-lactoglobulin is measured from me N-15 te~ s of me control peptide or B-lactoglobulin, respectively. That is, the amino acid residue at, for in~t~nçe~ residue location number 10 of the control peptide will be diLrerelll from the amino acid resi-hles located at residue loc~hon numbers 9-11 of ~-lactoglobulin. In this m~nner~ lag from residues that s~rigin~e~l from the control peptide will not inl~lrere with residues mat 20 origin~ted from ~-lactoglobulin, or vice versa, because at least one sequencer cycle will occur following the sequençing of a particular residue before that particular residue is sequenced again, whemer it origin~ted from the control peptide or ~-lactoglobulin. Useful non-interfering control peptides include, butare not limited to, peptides having the sequences given in SEQ ID NO:3, SEQ
25 ID NO:4 and SEQ ID NO:6.

CompositiQn~ cont~ining at least two of the control peptides c~e.~ed above are particularly useful for mo"ilo, ;.lg the proper operation of amino acid sequencers because they are capable of more quickly co,,r;. ..,;"g 30 whemer the system properly identifies all the pertinent amino acid resi~ es.
That is, me control peptides selected for use in the composition can be seqllenre~ im~llt~neously without data interference with each other. Further, wo94/o28s6 Pcr/uss3/o6932--~,140891 20 the peptides can be syntheci7ed to have complementary properties such that the amino acid residues located within 15 residues of the N-te~ us of one peptide are ~ençr~lly different from those located in the col,~sponding region on any other peptide. In this manner, it is possible to formul~te a composition which, S when subjected to sequencing, provides infor~tion regarding many, plefelably at least five, uncommon or ~lifficult to m~ ~lre amino acids, such as cysteine, ~gptophan, serine, threonine, histidine, ~nd arginine, within the first 15 cycles, ~ic~rably the first 10 cycles, of the sequencer. ~cltlition~lly, the control peptides can be ~lesigne~ to complement each other with respect to providing 10 inform~tion regarding the common amino acids, preferably having at least 8, ~rGfGlably at least 10, commc~n amino acids located within the first 15 amino acid res;d~les from the N-termini of the peptides. Fig. 6 shows the amino acid structure for SEQ ID N0:3 and SEQ ID N0:4 in addition to labeling the flifflr~ t amino acids located within 10 recidues of the N-termini.
As tlicclls~e~l above, preferably none of the peptides in the composition ;--lrl rere with B-lactoglobulin; furthermore, to allow measurement of lag and repetitive yield, none of the peptides in the composition should - r~re with each other. That is, each control peptide amino acid residue at a 20 particular residue loc~tic)n number differs from the amino acid residue for any other control peptide ha~ng a residue location number ranging from the particular residue loc~t-on number minus 1 to the particular residue location number plus 1. Useful compo~ition~ cont~ining at least two control peptides in~lutle, but are not limited to, compositions co~ the peptides having the 25 sequences given in SEQ ID N0:3 and SEQ ID N0:4 or SEQ ID N0:5 and SEQ
ID N0:6.

Small amounts, similar to amounts used to sequence unknowns, of the control peptide or a composition co-~ at least two control peptides, 30 preferably co.ll~illil-g a~roxilll~tely equal amounts of the peptides, can beplaced on the glass filter disc of an amino acid sequencer. If desired, B-lactoglobulin, preferably about 100 pmol, may also be placed on this filter. The ~ WO 94/02856 2 1 4 0 8 g 1 PCr/US93/06932 ?

sequencer ~-~tom~ti~-ally repeats a series of re~ct on~ as described above for the inte.rn~l st~n~rd peptide. Each cycle of the sequencer result should give the e~ecte~l synthetic PTH amino acids in an amount that is reasonable and reproducible when the equipment is operating normally. If the expected results 5 are not achieved, adjustments to the parameters controlling the amino acid seq~lencer, such as ~h~ngin~ the flow rates of the various reactants, washing times, drying times, injection vol~lmes, etc., can be made to optimize the aminoacid sequencer for peptide seq lerlcing.

As ~ se~l above, the synthetic control peptides of this invention are capable of ~-~sessing amino acid sequencer perforTn~nce by allowing the me~..cl~lent of repetitive yield, which is extremely important in de~el...i.-i.lg the operation of the sequencer. Knowledge regarding repetitive yield aids in the op~ lion process, which in turn illlpl~v~s the çh~nres for the proper 15 i~le~ n of ~1iffl~ult amino acids. Preferably, the repetitive yield should be detel...;~-P-l from m~ltiple occullGnces of a residue which delivili~es to a PTH-amino acid that is both stable and extracts well.

Repetitive yield values are commonly measured and averaged by 20 sequencing the l,ro~eill B-lactoglobulin. One disadvantage of using this protein, however, is that the sequencer is only opl;...;~e~l for high molecular weight components and does not insure that the equipment will work properly for relatively small peptides. For e~mple, e~essiv~ flow rates or washing times do not increase the amount of sample loss as much for ~-lactoglobulin as for low 25 molecular weight peptides, such as the small peptides that are normally obtained during int~.rn~l seqllen-~ing experime~ts~ Thus, too much solvent flow could wash the peptide off the support long before the C-lel"lh~us is reached. By opl;...;,il-g the sequencer using a control peptide of this invention, preferably a composition cont~ining a l~ ule of the control peptides, most preferably 30 simultaneously using a composition containing a mixture of the control peptides and B-lactoglobulin, it is possl~le to determine the optimi7~tion for both low and high molecular weight components. For instance, in a single 17 cycle experiment W094/02856 . ~ ~ Pcr/us93/06932 ~
21~0~9~

in which a composition CQl~t~ g a miYture of control peptides having the sequences of SEQ ID N0: 3 and SEQ ID N0:4 is simultaneously sequenced with ~-lactoglobulin, 7 and 3 good repetitive yield values, respectively, can beobtained. ~Mition~lly~ this procedure provides for the determin~tion of 3 initial 5 yields.

The synthetic control peptides of this invention can also be used as controls in a wide variety of chemical and enzymatic reactions. Successful microsequence analyses of samples available in limited q~l~ntities, or purified by 10 lD or 2D-PAGE, require precise l~tili7~tion of techniques and m~Yim~lly effiriont operation of all analytical systems. This is especially important whenintern~l seqse-n~ing on this type of sample is performed. Therfore, it is il~e,alive that c~-mic~l mo-lifir~on and cleavage re~rtionc and enzymatic digests are periodically tested or done in parallel with the sample unknowns.
15 This testing insures that the ~ec;l~d results are obtained or intlir~tes where problems may exist.

The control peptides are ~lesigned so that specific amino acid resi~lues are strategically placed within the amino acid sequences to provide 20 cleavage sites for these re~chon.c. Preferably, the control peptide contains amino acid patterns that are capable of reacting with at least 4, more preferably 5 ormore amino acid cleavage re~ct~ntc. The resulting fragments can then be analyzed to qualitatively and qu~ ;vcily assess that the desired cleavages occurred. For example, the cleavage products can be analyzed and purified by 25 HPLC and then all peaks identified by amino acid sequencing. Future experiments using the same cleavage conditions would also check the reproducibility of peptide retention times observed by HPLC analysis.

Thus, peptide or protein cleavage reactions can be monitored by 30 reacting a control peptide of this invention having at least one specific amino acid cleavage site with an amino acid cleavage reactant capable of cleaving a protein or peptide at the specific amino acid cleavage site; analyzing the WO 94/02856 2 1 4 0 8 9 1 PCI~US93/06932 cleavage products to determine the identity and quantity of the cleavage products; and cc,...p~ the identity and quantity of the cleavage products to the e~ecte~l yield for the re~ctinn between the control peptide and the amino acid cleavage re~ct~nt to monitor the peptide or protein cleavage reaction.
5 P~efellcd control peptides include SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. EYamples of common amino acid cleavage reactants in~ e, but are not limited to, Endoproteinase Asn-C, Endoproteinase Lys-C, Endoprote~lase Arg-C, EndoyIoteillase Glu-C and Endoproteinase Asp-N, which can be obtained from M~nnh~im Boehringer Biochemica (Indianapolis, 10 IN) and BNPS-~ tole, trypsin, cyanogen bromide, V8-E-AB (V8 protease which cleaves E in ammonium bicarbonate), V8-DE-PO~ (V8 protease specific for D
and E in phosphate buffer), formic acid and acetic acid.

The present control st~nd~rds are more particularly described in 15 the following eY~mples which are intended as illustrative only since numerousmo~lifir~tion~ and v~ri~tion~ therein will be a~palellt to those skilled in the art.

Example 3 The control peptides of this invention can be synthesi7ed, cleaved 20 and deprotected, and purified using the procedures outlined in Example 1 above for the synthesis of the internal standard peptide. The following synthetic amino acids, with the in(1ir~ted protecting groups, can be used for the synthesi~: Fmoc-nine; Fmoc-L-arginine (Pmc); Fmoc-L-asparagine (Trt); Fmoc-L-aspartic acid (OtBu); Fmoc-~cysteine (Trt); Fmoc-L-glut~...;.~e (Trt); Fmoc-~glutamic 25 acid (OtBu); Fmoc-L-glycine; Fmoc-L-histidine (Trt); Fmoc-L-isoleucine; Fmoc-L-leucine; Fmoc-L-lysine (Boc); Fmoc-L-methionine; Fmoc-L-phenyl~l~nine; F-moc-proline; Fmoc-L-serine (tBu); Fmoc-L-threonine (tBu); Fmoc-L-lly~tophan;
Fmoc-L~tyrosine (tBu); and Fmoc-L-valine (protecting group abbrevi~ffon.c: Boc = t-butylo~ye~lJonyl; Otbu = tert-butyl ester; Pmc = 2,2,5,7,8-pentamethyl-30 chroman-6-sulfonyl; tBu = tert-butyl; and Trt = trityl). Control peptides given by the sequences shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 were synthesized in this manner.

Wo 94/02856 ~ Pcr/us93/06932 --~2~4oa9~ 24 ExamPIe 4 Control peptides SEQ ID NO:3 and SEQ ID NO:4 were simultaneously sequenced as were SEQ ID NO:5 and SEQ ID NO:6 to s~lcces~ lly veriljr that these pairs of peptides were no~ telre~ g and were able to provide info~n~tion regarding ~lnrommon~ diffilcult to measure and common amino acid. Various supports can be used such as Problot (particularly for transblotted s~mples or samples purified from lD or 2D-PAGE experiments) or Porton Peptide filters (for s~mples purified by HPLC). Figure 8 shows typical HPLC chrom~tographic results from cycles 2, 5, 6, 7, 9 and 10 of the ~imlllt~npous seqnenrinE of SEQ ID NO:3 and SEQ ID NO:4, demonstrating the iAe~ ;c~l;on of iifficult to measure or llnrcmmon amino acids using these two control peptides.

Exsmple 5 Figs. 9 and 10 show examples of the theoretical results obtained from the re~rh~n of some commonly employed chemical and enzymatic amino acid cleavage re~ct~nt~ with the control peptides having the sequences shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. Fig. 11 shows the e~e~ ental co..fi~nl~hon of the allylation reaction shown in Fig. 9 for SEQ ID NO:3. (SEQ
20 ID NO:3 was inrllh~ted for 2 h at 60C in pH 8.6, O.5M Tris cont~ining 6M
nirline-HCl, 0.3% EDTA, 2% acetonitrile and a 50-fold molar excess of Dl'r. A 1.2 molar excess of ioclo~etic acid was then added and the solution incubated in the dark for 30 min.) Further co,.l i. ~ tion of the utility of the control peptides as controls for chemical and enzymatic amino acid cleavage reactions can be found in the experimental co~ n of the results predicted in Figs. 9 and 10. For in~t~nce, SEQ ID NO:4 is cleaved as predicted when reacted with Endoproteinase Asp-N (SEQ ID NO:4 was incubated at 37C for 24 h in ph 8.0, 0.05M sodium phosphate cont~ining 8% acetonitrile and a 1:20 (whv) ratio of ASp-N to peptide); formic acid (SEQ ID NO:4 was incubated in 75% formic WO 94/02856 21 4 08 g 1 PCr/US93/06932 acid for 5 days at 37C); and cyanogen bromide (SEQ ID NO:4 was incubated in 70% formic acid co..l~;..i..g 3% CNBr in the dark for 15 h).

Although the present process has been described with refelence to 5 specific details of certain embodiments thereof, it is not intended that such details should be regarded as limit~tions upon the scope of the invention exceptas and to the extent that they are inchl~led in the accompanying claims.

W O 94/02856 PC~r/US93/06932 -2 ~ ~08 g `1 .~ r SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: The Government of the United States of America, as represented by The Secretary (B) STREET: 6011 Executive Boulevard, Suite 325 (C) CITY: Rockville (D) STATE: Maryland (E) COUNTRY: United States of America (F) POSTAL CODE (ZIP): 20852 (G) TELEPHONE: 301/496-7056 (H) TELEFAX: 301/402-0220 (I) TELEX: None (ii) TITLE OF INVENTION: NOVEL AMINO ACID SEQUENCING
PEPTIDES AND METHODS FOR THEIR USE
(iii) NUMBER OF SEQUENCES: 6 (iv) COMPUTER READABLE FORM:
'A' MEDIUM TYPE: Floppy disk 'B; COMPUTER: IBM PC compatible C OPERATING SYSTEM: PC-DOS/MS-DOS
,D; SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: Not yet assigned (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/920,130 (B) FILING DATE: 24-JULY-1992 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
A' LENGTH: 28 amino acids B' TYPE: amino acid ,D, TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 94/02856 2 1 ~ 0 8 9 1 PCI~US93/06932 (ix) FEATURE:
,'A' NAME/KEY: Modified-site B LOCATION: 1..28 ;D, OTHER INFORMATION: /note= "Change all occurrences of Lys to Orn"
(ix) FEA-URE:
,A' NAME/KEY: Modified-site B LOCATION: 2..22 ,D; OTHER INFORMATION: /note= "Change all occurrences of Val to Nvl"
(ix) FEA-URE:
,'A' NAME/KEY: Modified-site ,B; LOCATION: 3..23 ;D; OTHER INFORMATION: /note= "Change all occurrences of Leu to Nle"
(ix) FEA-URE:
'A' NAME/KEY: Modified-site B LOCATION: 4..26 ,D, OTHER INFORMATION: /note= "Change all occurrences of Ala to Aab"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Lys Val Leu Ala Lys Val Ala Lys Val Ala Lys Val Leu Ala Lys Val Ala Lys Val Ala Lys Val Leu Lys Lys Ala Lys Lys (2) INFORMATION FOR SEQ ID NO:2:
(i) S:QJENCE CHARACTERISTICS:
A LENGTH: 35 amino acids ,B, TYPE: amino acid ;D, TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
'A' NAME/KEY: Modified-site B, LOCATION: 1..35 ;D, OTHER INFORMATION: /note= "Change all occurrences of Lys to Orn"
(ix) FEATURE:
A' NAME/KEY: Modified-site ,B, LOCATION: 2..30 ;D, OTHER INFORMATION: /note= "Change all occurrences of Leu to Nle"

W 0 94/028~6 ` PCT/US93/06932 2~4~89~(ix) F ATURE:
A' NAME/KEY: Modified-site B LOCATION: 3..29 ;D, OTHER INFORMATION: /note= "Change all occurrences of Val to Nvl"
(ix) FEATURE: ~
A' NAME/KEY: Moditfied-site B LOCATION: 4..33 ,D, OTHER INFORMATION: /note= "Change all occurrences of Ala to Aab"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Lys Leu Val Ala Lys Val Ala Lys Val Leu Ala Lys Val Ala Lys Val Ala Lys Val Leu Ala Lys Val Ala Lys Val Ala Lys Val Leu Lys Lys Ala Lys Lys (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQJENCE CHARACTERISTICS:
'A LENGTH: 27 amino acids B TYPE: amino acid ;D; TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Asp Cys Leu Lys Val Trp Gly Asp Ser Thr Lys Val Leu Glu Asn Arg Phe Tyr Leu Lys Ala Ile Arg Val His Leu Lys (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 27 amino acids 'B TYPE: amino acid ,D; TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~ W 0 94/02856 2 1 ~ 0 8 9 1 PC~r/US93/06932 29 ` ' xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: . , Lys Ala Glu Phe His Leu Arg Phe Glu Met Ala Arg Phe Asp Pro Leu Lys Ile Gln Phe Val Asp Lys Ala Tyr Phe Lys " 25 (2) INFORMATION FOR SEQ ID NO:5:
(i) S:QJENCE CHARACTERISTICS:
,A, LENGTH: 25 amino acids B TYPE: amino acid ;D, TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Val Leu Ile Val Trp Cys Asp Ser Thr Asn Leu Ile Val Gly Glu Gln Lys Tyr Ala Leu Lys Ile Val Gly Lys (2) ~NFORMATION FOR SEQ ID NO:6:
(i) S:QJENCE CHARACTERISTICS:
A LENGTH: 25 amino acids B TYPE: amino acid ,D; TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Phe Ala Tyr Phe His Leu Arg Phe Val Met Ala Tyr Phe Pro Leu Phe Lys Ile Val Phe Lys Ala Tyr Phe Lys

Claims (22)

1. An internal standard for amino-acid sequencing, comprising a peptide consisting essentially of unnatural amino-acid residues joined by amide linkages and having an amino-acid sequence containing at least two different unnatural amino-acid residues, wherein each unnatural amino-acid residue reacts with an amino-acid sequencer toyield a stable derivative with a retention time distinct from the corresponding retention times for natural amino-acid residues, and wherein two consecutive occurrences of at least one unnatural amino-acid residue in the amino-acid sequence are separated by at least one differing unnatural amino-acid residue.

2. An internal standard for amino-acid sequencing, comprising a peptide having at least five consecutive unnatural amino-acids joined by amide linkages, wherein the unnatural amino-acids contain at least two different residues and each unnaturalamino-acid residue reacts within an amino-acid sequencer to yield a stable derivative with a retention time distinct from the corresponding retention times for natural amino-acid residues, and wherein two consecutive occurrences of each unnatural amino-acid residue in the peptide are separated by at least one differing amino-acid residue.

3. The internal standard of claim 1 or claim 2, wherein the stable derivative is a phenylthiohydantoin derivative.

4. An internal standard for amino-acid sequencing, comprising a peptide consisting essentially of unnatural amino-acid residues joined by amide linkages and having an amino-acid sequence containing at least two different unnatural amino-acid residues, wherein each unnatural amino-acid residue reacts with an amino-acid sequencer toyield a stable derivative with a retention time distinct from the corresponding retention times for natural amino-acid residues, and wherein at least 70% of theunnatural amino-acid residues are positioned in the amino-acid sequence so as to be separated by at least one differing amino-acid residue.

5. An internal standard for amino-acid sequencing, comprising a peptide having at least five consecutive unnatural amino-acids joined by amide linkages, wherein the unnatural amino-acids contain at least two different residues and each unnaturalamino-acid residue reacts within an amino-acid sequencer to yield a stable derivative with a retention time distinct from the corresponding retention times for natural amino-acid residues, and wherein at least approximately 70% of the unnatural amino -acid residues are positioned in the peptide so as to be separated by at least one differing amino-acid residue.

6. The internal standard of any preceding claim, wherein the unnatural amino-acid residues are selected from ornithine, norvaline, norleucine and .alpha.-aminobutyric acid.

7. The internal standard of any preceding claim, wherein the amino-acid sequencecomprises 2 to 100 amino-acid residues in a single peptide chain.

8. The internal standard of any preceding claim, further comprising a charged substrate or a solid support attached at or near the C-terminus of the amino-acid sequence, wherein the charged substrate or solid support minimises wash-out of the internal standard from the amino-acid sequencer.

9. The internal standard of claim 8, wherein the charged substrate is selected from a peptide comprising at least one charged unnatural amino-acid residue, a peptide comprising at least one charged natural amino-acid residue, a peptide comprising a mixture of charged unnatural and charged natural amino-acid residues and a multiple antigenic peptide resin.

10. An internal standard for amino-acid sequencing consisting essentially of theamino-acid sequence of SEQ ID N0:1 or SEQ ID N0:2.

11. A method for monitoring the performance of an amino-acid sequencer during the sequencing of an unknown peptide or protein that is capable of distinguishing between a missed injection sequencer error and an error caused by faulty delivery of chemicals during the sequencer reactions, comprising the steps of:
(1) simultaneously sequencing the internal standard of any preceding claim and the unknown peptide or protein in the amino-acid sequencer to produce a chromatogram, wherein the retention times corresponding to the unnatural amino-acid residues of the internal standard are resolved from the retention times corresponding to natural amino-acids; and (2) comparing the retention times corresponding to the residues of the internal standard with predetermined information relating to the internal standard.

12. The method of claim 11, wherein two consecutive occurrences of at least one unnatural amino-acid residue in the amino-acid sequence of the internal standard are separated by at least one differing amino-acid residue, further comprising the step of determining the repetitive yield of the two consecutive occurrences.

13. The method of claim 11, wherein approximately 70% of the unnatural amino-acid residues in the internal standard are positioned in the amino-acid sequence so as to be separated by at least one differing amino-acid residue, further comprising the step of determining the lag corresponding to each separated unnatural amino-acidresidue.

14. A control peptide having an amino-acid sequence comprising from 3 to 100 natural amino-acid residues, wherein (1) the amino-acid sequence has at least 2 amino-acid residues selected from Cys, Trp, Ser, Thr, His, Arg and Met located within 15 amino-acid residues from the N-terminus of the peptide; and (2) each control peptide amino-acid residue at a particular residue location number differs from the .beta.-lactoglobulin amino-acid residue having a residue location number ranging from the particular residue location number minus 1 to the particular residue location number plus 1, wherein the residue location number for the control peptide is measured from its N-terminus and the residue location number for B-lactoglobulin is measured from its N-terminus.

15. The control peptide of claim 14, wherein the amino-acid sequence is selectedfrom SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5 and SEQ ID N0:6.

16. A composition comprising at least two control peptides that each have an amino-acid sequence comprising from 3 to 100 natural amino-acid residues, wherein (1) each control peptide has at least one of each of Cys, Trp, Ser, Thr, His and Arg residues within 15 amino-acid residues from the N-terminus of the control peptide; and (2) each control peptide amino-acid residue at a particular residue location number differs from the amino-acid residue for any other control peptide having a residue location number ranging from the particular residue location number minus 1 to the particular residue location number plus 1.

17. The composition of claim 16, wherein the amino-acid sequences of the at least two control peptides contain at least 20 different natural amino-acid residues.

18. The composition of claim 16, wherein the at least two control peptides comprise amino-acid sequences selected from SEQ ID N0:3, SEQ ID N0:4, SEQ ID
N0:5 and SEQ ID N0:6.

19. A method for monitoring the performance of an amino-acid sequencer to allow optimisation of the sequencer with respect to peptides, comprising the steps of:(1) sequencing at least one control peptide of claim 14 or claim 15 in the amino-acid sequencer to produce a chromatogram containing amino-acid residue information related to the retention times and intensities of the derivatised amino-acid residues contained in the at least one control peptide, the lag from one cycle of the amino-acid sequencer to another, and the repetitive yield for at least one amino-acid residue;
and (2) comparing the amino-acid residue information with predetermined information relating to the at least one control peptide.

20. The method of claim 19, further comprising the step of simultaneously sequencing .beta.-lactoglobulin with the at least one control peptide to produce ,.beta.-lactoglobulin residue information related to the retention times and intensities of the derivatised amino-acid residues contained in .beta.-lactoglobulin, the lag from one cycle of the amino-acid sequencer to another, and the repetitive yield for at least one amino-acid residue; and (2) comparing the .beta.-lactoglobulin residue information with predetermined information relating to .beta.-lactoglobulin.

21. A method for monitoring peptide or protein cleavage reactions, comprising the steps of:
(1) reacting the control peptide of claim 14 or claim 15 with an amino-acid cleavage reactant capable of cleaving a protein or peptide at a specific amino-acid cleavage site in the control peptide;
(2) analysing the cleavage products to determine the identity and quantity of the cleavage products; and (3) comparing the identity and quantity of the cleavage products to the expected yield for the reaction between the control peptide and the amino-acid cleavage reactant.

22. The method of claim 21, wherein the amino-acid cleavage reactant is selectedfrom Endoproteinase Asn-C, Endoproteinase Lys-C, Endoproteinase Arg-C, Endoproteinase Glu-C, Endoproteinase Asp-N, BNPS-skatole, trypsin, cyanogen bromide, V8-E-AB, V8-DE-PO4, formic acid and acetic acid.